Human interleukin-6 receptor expression inhibitor

ABSTRACT

The present invention discloses an expression inhibitor of human interleukin-6 receptor (human IL-6R) comprising as its active ingredient an antisense oligonucleotide derivative that hybridizes to a region of at least 9 to 30 consecutive nucleotide sequences that contains a nucleotide sequence of a portion being able to form a loop structure of mRNA that codes for human IL-6R.

TECHNICAL FIELD

The present invention relates to an antisense oligonucleotide derivativewhich is useful as a pharmaceutical that inhibits the expression ofhuman interleukin-6 receptor (IL-6R).

BACKGROUND ART

Human interleukin-6 (human IL-6) is a cytokine that was cloned as afactor that induces the final stage of differentiation of B cells toantibody-producing cells (Kishimoto, T. et al., Blood 74, 1-10, 1989).At present, it is known to have various effects, including induction ofacute phase protein in the liver (Kishimoto, T. et al, Blood 74, 1-10,1989).

In addition, IL-6 has been reported to be produced not only in lymphoidcells, but also in fibroblasts, vascular endothelial cells, urinarybladder carcinoma cell strain T24 and glioblastomas (Kohase, M. et al.,J. Cell Physio. 132, 271-278, 1978; Meir E. V. et al., Cancer Res. 50,6683-6688, 1990). Moreover, it also has a diverse range of target cells(Kishimoto, T. et al., Blood 74, 1-10, 1989).

In recent years, IL-6 has been reported to function as autocrine growthfactor in myeloma cells (Kawano, M. et al., Nature, 332, 83-85, 1988).Moreover, similar reports have been made with respect to nephrocytoma(Miki, S. et al., FEBS Letter 250, 607-610, 1989).

On the other hand, the signal for cell growth or differentiation byhuman IL-6 is known to be transmitted to cells by means of human IL-6Rand glycoprotein gp130 present on the cell surface (Taga, T. et al.,Cell 58, 573-581, 1989; Hibi, M. et al., Cell 68, 1149-1157, 1990).

In recent years, as a method for suppressing the function of a gene thatis the cause of a particular disease, the use of a oligonucleotidecomplementary to mRNA transcribed from DNA (antisense oligonucleotide)has been proposed to inhibit expression of said protein (Murakami,Chemistry 46, 681-684, 1991).

Moreover, as a means of eliminating problems such as the life, stabilityand rate of uptake into cells of antisense oligonucleotides, modifiedantisense oligonucleotides, such as methylphosphonate derivatives, inwhich the oxygen of a phosphate group of the nucleotide is substitutedwith a methyl group, and phosphorothioate derivatives, in which theoxygen group is substituted with sulfur (Murakami, Chemistry 46,681-684, 1991) are known. In actuality, these antisense oligonucleotideshave been observed to inhibit synthesis of viral protein (Agris, C. H.et al., Biochemistry, 25, 6268-6275, 1986).

Based on this concept, Levy, Y. et al. confirmed that the growth ofmyeloma cell strains, for which human IL-6 is a growth factor, isinhibited as a result of translation of mRNA for IL-6 being inhibited byantisense oligonucleotide (Levy, Y. et al., J. Clin. Invest., 88,696-699, 1991).

However, an antisense oligonucleotide derivative that significantlyinhibits expression of IL-6R in various cells in which IL-6R isexpressed is not known.

DISCLOSURE OF THE INVENTION

Thus, the present invention is intended to provide an antisenseoligonucleotide derivative that inhibits the expression of human IL-6R.

More specifically, the present invention provides a human IL-6Rexpression inhibitor comprising an antisense oligonucleotide derivativecorresponding to at least nine consecutive nucleotide sequences thatcontain the nucleotide sequence of the portion that has a highpossibility of being able to form a loop structure of mRNA that codesfor human IL-6R.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a graph indicating that the antisense oligonucleotidederivatives of the present invention in Example 1 (SEQ ID NOs: 2 and 3)inhibit expression of soluble IL-6R.

FIG. 2 is a graph indicating that the antisense oligonucleotidederivatives of the present invention in Example 1 (SEQ ID NOs: 2, 9 and10) inhibit expression of soluble IL-6R.

MODE FOR CARRYING OUT THE INVENTION

The inventors of the present invention searched for those nucleotidesequences considered to easily form a loop structure in terms of freeenergy and structural factors in human IL-6R mRNA using commerciallyavailable computer software for calculating free energy (e.g., Genetyx,Secondary Structure Search and Hairpin Loop-Stem Parts Searchmanufactured by Software Development Co., Ltd.). When an antisenseoligonucleotide derivative containing that loop structure wassynthesized, it was found to inhibit expression of human soluble IL-6R(sIL-6R), thereby leading to completion of the present invention.

In the preferable mode of the present invention, antisenseoligonucleotide derivatives corresponding to sequences of 9 to 30, andmore preferably, 12 to 25 consecutive nucleotides that contain anucleotide sequence being able to form a loop structure of mRNA thatcodes for human IL-6R.

The “antisense oligonucleotide” described here refers not only to thatin which all the nucleotides corresponding to nucleotides that form theprescribed region of DNA or mRNA are complementary, but also to that inwhich some mismatches are present provided the DNA or mRNA can be stablyhybridized with the oligonucleotide.

The nucleotide sequence of the cDNA for human IL-6R is as follows (see,for example, Japanese Unexamined Patent Publication No. 2-288,298 orScience, 241, 825-828 (1988): (SEQ ID NO: 1).

Of the nucleotide sequences, nucleotide sequences of the antisenseoligonucleotide derivative of the present invention are those which canbe suitably selected from sequences of consecutive nucleotide sequencesthat are able to form a loop structure found according to theabove-mentioned method.

Here, the portion that is able to form a loop structure of mRNA refersto the portion in which intramolecular hydrogen bonds of mRNA hardlyexist, and often exist as a single strand state.

More specifically, a portion that often exists as a single strand statecan be found, when the mRNA adopts a stable structure, using theabove-mentioned computer software.

In the present invention, examples of portions that are able to form theabove-mentioned loop structure in the nucleotide sequence shown in SEQID NO: 1 include around 640 to 685, 770 to 810, 1340 to 1375, 460 to475, 535 to 560 as well as around 925 to 960 and 815 to 840.

Thus, the oligonucleotide of the present invention is an oligonucleotidecomprising, for example, at least nine consecutive nucleotides in theabove-mentioned region.

According to one embodiment of the present invention, an expressioninhibiting oligonucleotide has the nucleotide sequence complementary tothe nucleotide sequence of the codons that codes for, for example, thesequence from Ala at position 75 to Leu at position 81 in SEQ ID NO: 1,namely 5′-AGCCTCCTTCCCATGCCAGC-3′ (SEQ ID NO: 2).

Other examples include an oligonucleotides having a nucleotide sequencecomplementary to the nucleotide sequence of the codons that code for thesequence from Leu at position 127 to Glu at position 133, namely5′-CTCACAAACAACATTGCTGA-3′ (SEQ ID NO: 3), that having a nucleotidesequence complementary to the nucleotide sequence of the codons thatcode for the sequence from His at position 70 to Met at position 77,namely 5′-TGCCAGCCCATCTGCTGGGG-3′ (SEQ ID NO: 4), that having anucleotide sequence complementary to the nucleotide sequence of thecodons that code for the sequence from Val at position 34 to Asp atposition 41, namely 5′-CTCCTGGCAGACTGGTCAGC-3′ (SEQ ID NO: 5), thathaving a nucleotide sequence complementary to the nucleotide sequence ofthe codons that code for the sequence from Gln at position 118 to Lys atposition 124, namely 5′-TTCCGGAAGCAGGAGAGCTG-3′ (SEQ ID NO: 6), thathaving a nucleotide sequence complementary to the nucleotide sequence ofthe codons that code for Pro at position 164 to Glu at position 170,namely 5′-TCCTGGGAATACTGGCACGG-3′ (SEQ ID NO: 7), that having anucleotide sequence complementary to the nucleotide sequence of thecodons that code for the sequence from Ala at position 75 to Leu atposition 82, namely 5′-GCAGCCTCCTTCCCATGCCA-3′ (SEQ ID NO: 9), and thathaving a nucleotide sequence complementary to the nucleotide sequence ofthe codons that code for the sequence from Trp at position 74 to Arg atposition 80, namely 5′-CTCCTTCCCATGCCAGCCCA-3′ (SEQ ID NO: 10).

The structure in the case wherein the oligonucleotide derivative used inthe present invention is a deoxyribonucleotide is as shown in ChemicalStructure 1 wherein X may independently be oxygen (O), sulfur (S), alower alkyl group or a primary or secondary amine. Y may independentlybe either oxygen (O) or sulfur (S). B is selected from among eitheradenine, guanine, thymine or cytosine, and is usually an oligonucleotidecomplementary to DNA or mRNA that codes for human IL-6 receptor. R isindependently hydrogen, a dimethoxytrityl group or a lower alkyl group.n is an integer from 7 to 28.

Preferable examples of oligonucleotide derivatives include not onlyunmodified oligonucleotides, but also modified oligonucleotides.Examples of these modified forms include the above-mentionedmethylphosphonate type or ethylphosphonate type of loweralkylphosphonate-modified forms, other phosphorothioate-modified formsand phosphoramidate modified forms.

These oligonucleotide derivatives can be obtained by conventionalmethods as described below.

The oligonucleotide in which X and Y are O in the Chemical Structure 1can be easily synthesized by a commercially available DNA synthesizer(e.g., that manufactured by Applied Biosystems).

Examples of synthesis methods that can be used to obtain thisoligonucleotide include solid phase synthesis using phosphoroamidite andsolid phase synthesis using hydrogen phosphonate.

For example, see T. Atkinson, M. Smith, in Oligonucleotide Synthesis: APractical Approach, ed. M. J. Gait, IRL Press, 35-81 (1984); M. H.Caruthers, Science, 230, 281 (1985); A. Kume, M. Fujii, M. Sekine, M.Hata, J. Org. Chem., 49, 2139 (1984); B. C. Froehler, M. Matteucci,Tetrahedron Lett., 27, 469 (1986); P. J. Garegg, I. Lindh, T. Regberg,J. Stawinski, R. Stromberg, C. Henrichson, ibid., 27, 4051 (1986); B. S.Sproat, M. J. Gait, in Oligonucleotide Synthesis: A Practical Approach,ed. M. J. Gait, IRL Press, 83-115 (1984); S. L. Beaucage and M. H.Caruthers, Tetrahedron Lett., 22, 1859-1862 (1981); M. D. Matteucci andM. H. Caruthers, Tetrahedron Lett., 21, 719-722 (1980); M. D. Matteucciand M. H. Caruthers, J. Am. Chem. Soc., 103, 3185-3191 (1981).

Phosphate triester-modified forms, in which X is a lower alkoxy group,can be obtained by conventional methods, an example of which is treatedan oligonucleotide obtained in chemical synthesis with a tosylchloridesolution of DMF, methanol and 2,6-lutidine (Moody, H. M., et al.,Nucleic Acids Res., 17, 4769-4782 (1989)).

Alkylphosphonate-modified forms, in which X is an alkyl group, can beobtained using conventional methods, an example of which usesphosphoramidite (M. A. Dorman, et al., Tetrahedron, 40, 95-102 (1984);K. L. Agarwal and F. Riftina, Nucleic Acids Res., 6, 3009-3024 (1979)).

Phosphorothiate-modified forms, in which X is S, can be obtained byconventional methods, examples of which include solid phase synthesisusing sulfur (C. A. Stein, et al., Nucleic Acids Res., 16, 3209-3221(1988), or solid phase synthesis using tetraethylthiuram disulfide (H.Vu and B. L. Hirschbein, Tetrahedron Letters, 32, 3005-3008 (1991)).

Phosphorodithioate-modified forms, in which X and Y are both S, can beobtained by, for example, solid phase synthesis by converting bisamiditeto thioamidite and reacting with sulfur (W. K.-D. Brill, et al., J. Am.Chem. Soc., 111, 2321-2322 (1989)).

Phosphoramidate-modified forms, in which X is a primary or secondaryamine, can be obtained by, for example, solid phase synthesis bytreating hydrogen phosphonate with a primary or secondary amine (B.Froehler, et al., Nucleic Acids Res., 16, 4831-4839 (1988).Alternatively, the above-mentioned modified forms can also be obtainedby oxidizing amidite with tert-butylhydroperoxide (H. Ozaki, et al.,Tetrahedron Lett., 30, 5899-5902 (1989)).

Purification and confirmation of purity can be performed withhigh-performance liquid chromatography or polyacrylamide gelelectrophoresis. Confirmation of molecular weight can be performed withelectrospray ionization mass spectrometry or fast atom bombardment massspectrometry. The antisense oligonucleotide derivative of the presentinvention may be synthesized in any manner and be of any origin providedit has a sequence that hybridizes with the nucleotide sequence of DNA ormRNA that codes for human IL-6R.

The antisense oligonucleotide derivative of the present invention actson human IL-6R producing cells so that the derivative bonds to DNA ormRNA that codes for human IL-6R so as to inhibit its transcription ortranslation, and ultimately resulting in suppressing the effect of humanIL-6 as a result of inhibiting expression of human IL-6R. Examples ofthe effects of human IL-6 inhibited by the antisense oligonucleotidederivative of the present invention include platelet proliferationeffects, antibody production enhancing effects, acute phase proteininducing effects, tumor cell growth effects and neuron differentiationeffects.

Thus, the antisense oligonucleotide derivative of the present inventionis considered to be effective in the treatment of diseases caused bythese effects, examples of which include carcinomas such as kidneycancer, myeloma, Lennert's T lymphoma and Kaposi's sarcoma, autoimmunediseases such as chronic rheumatoid arthritis, mesangium proliferativenephritis, psoriasis, carcinomatous cachexia and endotoxin shock ininfections.

The antisense oligonucleotide derivative of the present invention can bemixed with suitable carriers that are inactive relative to saidderivative to prepare external preparations such as liniments andpoultices.

In addition, the antisense oligonucleotide derivative of the presentinvention can also be formulated to tablets, powders, granules,capsules, liposome capsules, injections, liquids, nasal drops as well asfreeze-dried products by adding vehicles, isotonic agents, solubilityassistants, stabilizers, preservatives or analgesics. These preparationscan be prepared in accordance with conventional methods.

The antisense oligonucleotide derivative of the present invention can beapplied either directly to the afflicted area on the patient, oreventually reach the afflicted area by intravenous administration and soforth. Moreover, antisense inclusion materials can also be used toimprove duration and membrane permeability. Examples of these includeliposomes, poly-L-lysine, lipids, cholesterol, lipofectin and theirderivatives.

The dose of the antisense oligonucleotide derivative of the presentinvention can be suitably adjusted according to the status of thepatient, and used in a preferable amount. For example, it can beadministered at a dose within the range of 0.1 to 100 mg/kg, andpreferably within the range of 0.1 to 50 mg/kg.

The following provides a detailed explanation of the present inventionthrough its embodiments.

EXAMPLES Synthesis Example 1 Synthesis of 5′-AGCCTCCTTCCCATGCCAGC-3′(SEQ ID NO: 2) (Phosphorothioate-modified Form)

The methoxytrityl group of 5′-dimethoxytrityl-2′-deoxycytidine, in whichthe 3′-hydroxy group was bonded to the support medium, was deprotectedby trichloroacetate, and 5′-dimethoxytrityl-2′-deoxyguanosineβ-cyanoethylphosphoramidite was condensed with its 5′-hydroxy group bytetrazole. After sulfiding the phosphorous with tetraethylthiuramdisulfide, the unreacted 5′-hydroxy group was acetylated with aceticanhydride and dimethylaminopyridine.

Deprotection, condensation, sulfidation and acetylation were repeated ina similar manner. The final 5′-dimethoxytrityl-2′-deoxyadenosineβ-cyanoethylphosphoramidite derivative was condensed and the resulting20-mer phosphorothioate-modified form following sulfidation (theabove-mentioned processes were performed with the Model 381A DNAsynthesizer manufactured by Applied Biosystems) was separated from thesupport medium with 2 ml of concentrated aqueous ammonia along withremoving the cyanoethyl group from the phosphorous and additionallyremoving the protection groups attached to adenine, guanine andcytosine.

The 5′-dimethoxytrityl protection group was removed from the resulting5′-dimethoxytrityloligonucleotide phosphorothioate in its original formwithout refining, or after purifying by high-performance liquidchromatography, or after retaining on a cartridge column forpurification of synthetic DNA (e.g., Oligopack SP manufactured by JapanMillipore). The resulting oligonucleotide phosphorothioate was purifiedwith high-performance liquid chromatography as necessary to obtainapproximately 1.37 mg of the target 5′-AGCCTCCTTCCCATGCCAGC-3′ (SEQ IDNO: 2) (phosphorothioate modified form).

Synthesis Example 2 Synthesis of 5′-CTCACAAACAACATTGCTGA-3′ (SEQ ID NO:3) (Phosphorothioate-modified Form)

Approximately 0.65 mg of the target 5′-CTCACAAACAACATTGCTGA-3′ (SEQ IDNO: 3) (phosphorothioate-modified form) were obtained in the same manneras Synthesis Example 1.

Synthesis Example 3 Synthesis of 5′-TGCCAGCCCATCTGCTGGGG-3′ (SEQ ID NO:4) (Phosphorothioate-modified Form)

Approximately 0.98 mg of the target 5′-TGCCAGCCCATCTGCTGGGG-3′ (SEQ IDNO: 4) (phosphorothioate-modified form) were obtained in the same manneras Synthesis Example 1.

Synthesis Example 4 Synthesis of 5′-CTCCTGGCAGACTGGTCAGC-3′ (SEQ ID NO:5) (Phosphorothioate-modified Form)

Approximately 1.61 mg of the target 5′-CTCCTGGCAGACTGGTCAGC-3′ (SEQ IDNO: 5) (phosphorothioate-modified form) were obtained in the same manneras Synthesis Example 1.

Synthesis Example 5 Synthesis of 5′-TTCCGGAAGCAGGAGAGCTG-3′ (SEQ ID NO:6) (Phosphorothioate-modified Form)

Approximately 1.47 mg of the target 5′-TTCCGGAAGCAGGAGAGCTG-3′ (SEQ IDNO: 6) (phosphorothioate-modified form) were obtained in the same manneras Synthesis Example 1.

Synthesis Example 6 Synthesis of 5′-TCCTGGGAATACTGGCACGG-3′ (SEQ ID NO:7) (Phosphorothioate-modified Form)

Approximately 1.64 mg of the target 5′-TCCTGGGAATACTGGCACGG-3′ (SEQ IDNO: 7) (phosphorothioate-modified form) were obtained in the same manneras Synthesis Example 1.

Synthesis Example 7 Synthesis of 5′-GCAGCCTCCTTCCCATGCCA-3′ (SEQ ID NO:9) (Phosphorothioate-modified Form)

Approximately 2.47 mg of the target 5′-GCAGCCTCCTTCCCATGCCA-3′ (SEQ IDNO: 9) (phosphorothioate-modified form) were obtained in the same manneras Synthesis Example 1.

Synthesis Example 8 Synthesis of 5′-CTCCTTCCCATGCCAGCCCA-3′ (SEQ ID NO:10) (Phosphorothioate-modified Form)

Approximately 2.06 mg of the target 5′-CTCCTTCCCATGCCAGCCCA-3′ (SEQ IDNO: 10) (phosphorothioate-modified form) were obtained in the samemanner as Synthesis Example 1.

Reference Example 1 Synthesis of 5′-CCCCAGCAGATGGGCTGGCA-3′ (SEQ ID NO:8) (Phosphorothioate-modified Form: Sense Sequence of SEQ ID NO: 4)

Approximately 0.53 mg of the target 5′-CCCCAGCAGATGGGCTGGCA-3′ (SEQ IDNO: 8) (phosphorothioate-modified form) were obtained in the same manneras Synthesis Example 1.

Reference Example 2 Synthesis of 5′-GCTGGCATGGGAAGGAGGCT-3′ (SEQ ID NO:11) (Phosphorothioate-modified Form: Sense Sequence of SEQ ID NO: 2)

Approximately 1.91 mg of the target 5′-GCTGGCATGGGAAGGAGGCT-3′ (SEQ IDNO: 11) (phosphorothioate-modified form) were obtained in the samemanner as Synthesis Example 1.

Experiment 1 Inhibitory Effect on Expression of Human Soluble IL-6R(sIL-6R)

(1) Preparation of CHO.SR344 Cells

pBSF2R.236 (Science, 241, 825-828 (1988)) was cleaved with SphI, and the1205 bp IL-6R cDNA fragment was inserted into mp18 (Amersham). sIL-6RcDNA was prepared by preparing a synthetic oligonucleotide of5′-ATATTCTAGAGAGCTTCT-3′ and using this oligonucleotide in in-vitromutagenesis system (Amersham). As a result, the termination codon wasthe 345th of the amino acid sequence.

dhfr-cDNA was inserted into the PvuII site of plasmid pECE (Cell, 45,721-735 (1986)) to prepare plasmid pECEdhfr. The HindIII-SalI fragmentof sIL-6R was inserted into plasmid pECEdhfr to prepare soluble IL-6Rexpression vector plasmid pECEdhfr344.

pECEdhfr344 was introduced into dhfr-CHO cells DXB-11 (Pro. Natl. Acad.Sci. U.S.A., 77, 4216-4220 (1980)) according to the calcium phosphatemethod, and amplified with MTX. Finally, 200 nM MTX-resistantsIL-6R-producing CHO cells (CHO.SR344) were prepared (J. Biochem., 108,673-676 (1990)). Ordinary culturing of the cells was performed in IMDMmedium (Gibco) containing 5% FCS (Xavier Investments) and 200 nM MTX.

(2) Effect of IL-6R Antisense Oligonucleotides on sIL-6R Production ofCHO.SR344 Cells

The CHO.SR344 cells were peeled from the culture dish with trypsin-EDTA(Gibco). After washing with culture liquid, the cells were additionallywashed with Non-Serum (trade name) serum-free medium and suspended in,Non-Serum, serum-free medium containing 200 nM MTX. 100 μl of CHO.SR344cell suspension (5×10⁴ cells/ml) and 100 μl of 2 μM IL-6R antisenseoligonucleotide were added to a 96-well culture plate followed byculturing in an incubator at 37° C. and 5% CO₂.

After culturing for 24 hours, the amount of soluble IL-6R in the culturesupernatant was measured by sandwich ELISA using mouse anti-IL-6Rmonoclonal antibody (MT18) (Japanese Unexamined Patent Publication No.2-288898) and rabbit anti-IL-6R polyclonal antibody. Furthermore, thesense oligonucleotide derivative to SEQ ID NO: 4 (SEQ ID NO: 8) or thesense oligonucleotide to SEQ ID NO: 2 (SEQ ID NO: 11) were measured ascontrols.

The human IL-6R antisense oligonucleotide exhibited inhibitory effectson expression of soluble IL-6R (FIGS. 1 and 2).

11 3319 base pairs nucleic acid single linear cDNA CDS 438..1844 1GGCGGTCCCC TGTTCTCCCC GCTCAGGTGC GGCGCTGTGG CAGGAAGCCA CCCCCTCGGT 60CGGCCGGTGC GCGGGGCTGT TGCGCCATCC GCTCCGGCTT TCGTAACCGC ACCCTGGGAC 120GGCCCAGAGA CGCTCCAGCG CGAGTTCCTC AAATGTTTTC CTGCGTTGCC AGGACCGTCC 180GCCGCTCTGA GTCATGTGCG AGTGGGAAGT CGCACTGACA CTGAGCCGGG CCAGAGGGAG 240AGGAGCCGAG CGCGGCGCGG GGCCGAGGGA CTCGCAGTGT GTGTAGAGAG CCGGGCTCCT 300GCGGATGGGG GCTGCCCCCG GGGCCTGAGC CCGCCTGCCC GCCCACCGCC CCGCCCCGCC 360CCTGCCACCC CTGCCGCCCG GTTCCCATTA GCCTGTCCGC CTCTGCGGGA CCATGGAGTG 420GTAGCCGAGG AGGAAGC ATG CTG GCC GTC GGC TGC GCG CTG CTG GCT GCC 470 MetLeu Ala Val Gly Cys Ala Leu Leu Ala Ala 1 5 10 CTG CTG GCC GCG CCG GGAGCG GCG CTG GCC CCA AGG CGC TGC CCT GCG 518 Leu Leu Ala Ala Pro Gly AlaAla Leu Ala Pro Arg Arg Cys Pro Ala 15 20 25 CAG GAG GTG GCA AGA GGC GTGCTG ACC AGT CTG CCA GGA GAC AGC GTG 566 Gln Glu Val Ala Arg Gly Val LeuThr Ser Leu Pro Gly Asp Ser Val 30 35 40 ACT CTG ACC TGC CCG GGG GTA GAGCCG GAA GAC AAT GCC ACT GTT CAC 614 Thr Leu Thr Cys Pro Gly Val Glu ProGlu Asp Asn Ala Thr Val His 45 50 55 TGG GTG CTC AGG AAG CCG GCT GCA GGCTCC CAC CCC AGC AGA TGG GCT 662 Trp Val Leu Arg Lys Pro Ala Ala Gly SerHis Pro Ser Arg Trp Ala 60 65 70 75 GGC ATG GGA AGG AGG CTG CTG CTG AGGTCG GTG CAG CTC CAC GAC TCT 710 Gly Met Gly Arg Arg Leu Leu Leu Arg SerVal Gln Leu His Asp Ser 80 85 90 GGA AAC TAT TCA TGC TAC CGG GCC GGC CGCCCA GCT GGG ACT GTG CAC 758 Gly Asn Tyr Ser Cys Tyr Arg Ala Gly Arg ProAla Gly Thr Val His 95 100 105 TTG CTG GTG GAT GTT CCC CCC GAG GAG CCCCAG CTC TCC TGC TTC CGG 806 Leu Leu Val Asp Val Pro Pro Glu Glu Pro GlnLeu Ser Cys Phe Arg 110 115 120 AAG AGC CCC CTC AGC AAT GTT GTT TGT GAGTGG GGT CCT CGG AGC ACC 854 Lys Ser Pro Leu Ser Asn Val Val Cys Glu TrpGly Pro Arg Ser Thr 125 130 135 CCA TCC CTG ACG ACA AAG GCT GTG CTC TTGGTG AGG AAG TTT CAG AAC 902 Pro Ser Leu Thr Thr Lys Ala Val Leu Leu ValArg Lys Phe Gln Asn 140 145 150 155 AGT CCG GCC GAA GAC TTC CAG GAG CCGTGC CAG TAT TCC CAG GAG TCC 950 Ser Pro Ala Glu Asp Phe Gln Glu Pro CysGln Tyr Ser Gln Glu Ser 160 165 170 CAG AAG TTC TCC TGC CAG TTA GCA GTCCCG GAG GGA GAC AGC TCT TTC 998 Gln Lys Phe Ser Cys Gln Leu Ala Val ProGlu Gly Asp Ser Ser Phe 175 180 185 TAC ATA GTG TCC ATG TGC GTC GCC AGTAGT GTC GGG AGC AAG TTC AGC 1046 Tyr Ile Val Ser Met Cys Val Ala Ser SerVal Gly Ser Lys Phe Ser 190 195 200 AAA ACT CAA ACC TTT CAG GGT TGT GGAATC TTG CAG CCT GAT CCG CCT 1094 Lys Thr Gln Thr Phe Gln Gly Cys Gly IleLeu Gln Pro Asp Pro Pro 205 210 215 GCC AAC ATC ACA GTC ACT GCC GTG GCCAGA AAC CCC CGC TGG CTC AGT 1142 Ala Asn Ile Thr Val Thr Ala Val Ala ArgAsn Pro Arg Trp Leu Ser 220 225 230 235 GTC ACC TGG CAA GAC CCC CAC TCCTGG AAC TCA TCT TTC TAC AGA CTA 1190 Val Thr Trp Gln Asp Pro His Ser TrpAsn Ser Ser Phe Tyr Arg Leu 240 245 250 CGG TTT GAG CTC AGA TAT CGG GCTGAA CGG TCA AAG ACA TTC ACA ACA 1238 Arg Phe Glu Leu Arg Tyr Arg Ala GluArg Ser Lys Thr Phe Thr Thr 255 260 265 TGG ATG GTC AAG GAC CTC CAG CATCAC TGT GTC ATC CAC GAC GCC TGG 1286 Trp Met Val Lys Asp Leu Gln His HisCys Val Ile His Asp Ala Trp 270 275 280 AGC GGC CTG AGG CAC GTG GTG CAGCTT CGT GCC CAG GAG GAG TTC GGG 1334 Ser Gly Leu Arg His Val Val Gln LeuArg Ala Gln Glu Glu Phe Gly 285 290 295 CAA GGC GAG TGG AGC GAG TGG AGCCCG GAG GCC ATG GGC ACG CCT TGG 1382 Gln Gly Glu Trp Ser Glu Trp Ser ProGlu Ala Met Gly Thr Pro Trp 300 305 310 315 ACA GAA TCC AGG AGT CCT CCAGCT GAG AAC GAG GTG TCC ACC CCC ATG 1430 Thr Glu Ser Arg Ser Pro Pro AlaGlu Asn Glu Val Ser Thr Pro Met 320 325 330 CAG GCA CTT ACT ACT AAT AAAGAC GAT GAT AAT ATT CTC TTC AGA GAT 1478 Gln Ala Leu Thr Thr Asn Lys AspAsp Asp Asn Ile Leu Phe Arg Asp 335 340 345 TCT GCA AAT GCG ACA AGC CTCCCA GTG CAA GAT TCT TCT TCA GTA CCA 1526 Ser Ala Asn Ala Thr Ser Leu ProVal Gln Asp Ser Ser Ser Val Pro 350 355 360 CTG CCC ACA TTC CTG GTT GCTGGA GGG AGC CTG GCC TTC GGA ACG CTC 1574 Leu Pro Thr Phe Leu Val Ala GlyGly Ser Leu Ala Phe Gly Thr Leu 365 370 375 CTC TGC ATT GCC ATT GTT CTGAGG TTC AAG AAG ACG TGG AAG CTG CGG 1622 Leu Cys Ile Ala Ile Val Leu ArgPhe Lys Lys Thr Trp Lys Leu Arg 380 385 390 395 GCT CTG AAG GAA GGC AAGACA AGC ATG CAT CCG CCG TAC TCT TTG GGG 1670 Ala Leu Lys Glu Gly Lys ThrSer Met His Pro Pro Tyr Ser Leu Gly 400 405 410 CAG CTG GTC CCG GAG AGGCCT CGA CCC ACC CCA GTG CTT GTT CCT CTC 1718 Gln Leu Val Pro Glu Arg ProArg Pro Thr Pro Val Leu Val Pro Leu 415 420 425 ATC TCC CCA CCG GTG TCCCCC AGC AGC CTG GGG TCT GAC AAT ACC TCG 1766 Ile Ser Pro Pro Val Ser ProSer Ser Leu Gly Ser Asp Asn Thr Ser 430 435 440 AGC CAC AAC CGA CCA GATGCC AGG GAC CCA CGG AGC CCT TAT GAC ATC 1814 Ser His Asn Arg Pro Asp AlaArg Asp Pro Arg Ser Pro Tyr Asp Ile 445 450 455 AGC AAT ACA GAC TAC TTCTTC CCC AGA TAG CTGGCTGGGT GGCACCAGCA 1864 Ser Asn Thr Asp Tyr Phe PhePro Arg * 460 465 GCCTGGACCC TGTGGATGAC AAAACACAAA CGGGCTCAGC AAAAGATGCTTCTCACTGCC 1924 ATGCCAGCTT ATCTCAGGGG TGTGCGGCCT TTGGCTTCAC GGAAGAGCCTTGCGGAAGGT 1984 TCTACGCCAG GGGAAAATCA GCCTGCTCCA GCTGTTCAGC TGGTTGAGGTTTCAAACCTC 2044 CCTTTCCAAA TGCCCAGCTT AAAGGGGTTA GAGTGAACTT GGGCCACTGTGAAGAGAACC 2104 ATATCAAGAC TCTTTGGACA CTCACACGGA CACTCAAAAG CTGGGCAGGTTGGTGGGGGC 2164 CTCGGTGTGG AGAAGCGGCT GGCAGCCCAC CCCTCAACAC CTCTGCACAAGCTGCACCCT 2224 CAGGCAGGTG GGATGGATTT CCAGCCAAAG CCTCCTCCAG CCGCCATGCTCCTGGCCCAC 2284 TGCATCGTTT CATCTTCCAA CTCAAACTCT TAAAACCCAA GTGCCCTTAGCAAATTCTGT 2344 TTTTCTAGGC CTGGGGACGG CTTTTACTTA AACGCCAAGG CCTGGGGGAAGAAGCTCTCT 2404 CCTCCCTTTC TTCCCTACAG TTCAAAAACA GCTGAGGGTG AGTGGGTGAATAATACAGTA 2464 TGTCAGGGCC TGGTCGTTTT CAACAGAATT ATAATTAGTT CCTCATTAGCAGTTTTGCCT 2524 AAATGTGAAT GATGATCCTA GGCATTTGCT GAATACAGAG GCAACTGCATTGGCTTTGGG 2584 TTGCAGGACC TCAGGTGAGA AGCAGAGGAA GGAGAGGAGA GGGGCACAGGGTCTCTACCA 2644 TCCCCTGTAG AGTGGGAGCT GAGTGGGGGA TCACAGCCTC TGAAAACCAATGTTCTCTCT 2704 TCTCCACCTC CCACAAAGGA GAGCTAGCAG CAGGGAGGGC TTCTGCCATTTCTGAGATCA 2764 AAACGGTTTT ACTGCAGCTT TGTTTGTTGT CAGCTGAACC TGGGTAACTAGGGAAGATAA 2824 TATTAAGGAA GACAATGTGA AAAGAAAAAT GAGCCTGGCA AGAATGCGTTTAAACTTGGT 2884 TTTTAAAAAA CTGCTGACTG TTTTCTCTTG AGAGGGTGGA ATATCCAATATTCGCTGTGT 2944 CAGCATAGAA GTAACTTACT TAGGTGTGGG GGAAGCACCA TAACTTTGTTTAGCCCAAAA 3004 CCAAGTCAAG TGAAAAAGGA GGAAGAGAAA AAATATTTTC CTGCCAGGCATGGAGGCCCA 3064 CGCACTTCGG GAGGTCGAGG CAGGAGGATC ACTTGAGTCC AGAAGTTTGAGATCAGCCTG 3124 GGCAATGTGA TAAAACCCCA TCTCTACAAA AAGCATAAAA ATTAGCCAAGTGTGGTAGAG 3184 TGTGCCTGAA GTCCCAGATA CTTGGGGGGC TGAGGTGGGA GGATCTCTTGAGCCTGGGAG 3244 GTCAAGGCTG CAGTGAGCCG AGATTGCACC ACTGCACTCC AGCCTGGGGTGACAGAGCAA 3304 GTGAGACCCT GTCTC 3319 20 base pairs nucleic acid singlelinear DNA (genomic) 2 AGCCTCCTTC CCATGCCAGC 20 20 base pairs nucleicacid single linear DNA (genomic) 3 CTCACAAACA ACATTGCTGA 20 20 basepairs nucleic acid single linear DNA (genomic) 4 TGCCAGCCCA TCTGCTGGGG20 20 base pairs nucleic acid single linear DNA (genomic) 5 CTCCTGGCAGACTGGTCAGC 20 20 base pairs nucleic acid single linear DNA (genomic) 6TTCCGGAAGC AGGAGAGCTG 20 20 base pairs nucleic acid single linear DNA(genomic) 7 TCCTGGGAAT ACTGGCACGG 20 20 base pairs nucleic acid singlelinear DNA (genomic) 8 CCCCAGCAGA TGGGCTGGCA 20 20 base pairs nucleicacid single linear DNA (genomic) 9 GCAGCCTCCT TCCCATGCCA 20 20 basepairs nucleic acid single linear DNA (genomic) 10 CTCCTTCCCA TGCCAGCCCA20 20 base pairs nucleic acid single linear DNA (genomic) 11 GCTGGCATGGGAAGGAGGCT 20

What is claimed is:
 1. An inhibitor of expression of human IL-6 receptorcomprising an optionally modified antisense oligonucleotide wherein saidantisense oligonucleotide has a nucleotide sequence selected from thegroup consisting of CTCACAAACA ACATTGCTGA (SEQ ID NO:3), CTCCTGGCAGACTGGTCAGC (SEQ ID NO:5), TTCCGGAAGC AGGAGAGCTG (SEQ ID NO:6), andTCCTGGGAAT ACTGGCACGG (SEQ ID NO:7).
 2. An inhibitor of expression ofhuman IL-6 receptor comprising an antisense oligonucleotide wherein saidantisense oligonucleotide has a nucleotide sequence selected from thegroup consisting of CTCACAAACA ACATTGCTGA (SEQ ID NO:3), CTCCTGGCAGACTGGTCAGC (SEQ ID NO:5), TTCCGGAAGC AGGAGAGCTG (SEQ ID NO:6), andTCCTGGGAAT ACTGGCACGG (SEQ ID NO:7).
 3. The inhibitor of claim 2,wherein said antisense oligonucleotide has a nucleotide sequenceCTCACAAACA ACATTGCTGA (SEQ ID NO:3).
 4. The inhibitor of claim 2,wherein said antisense oligonucleotide has a nucleotide sequenceCTCCTGGCAG ACTGGTCAGC (SEQ ID NO:5).
 5. The inhibitor of claim 2,wherein said antisense oligonucleotide has a nucleotide sequenceTTCCGGAAGC AGGAGAGCTG (SEQ ID NO:6).
 6. The inhibitor of claim 2,wherein said antisense oligonucleotide has a nucleotide sequenceTCCTGGGAAT ACTGGCACGG (SEQ ID NO:7).
 7. An inhibitor of expression ofhuman IL-6 receptor comprising an antisense oligonucleotide, saidantisense oligonucleotide having the structure:

wherein X and X′ are independently sulfur, a lower alkyl group, aprimary amine, a secondary amine, or a lower alkoxy; Y and Y′ areindependently oxygen or sulfur; B, B′ and B″ are independently adenine,guanine, thymine or cytosine; R and R′ are independently hydrogen,dimethoxytrityl or a lower alkyl group; and n is from 7 to 28, furtherwherein said antisense oligonucleotide has a nucleotide sequenceselected from the group consisting of: CTCACAAACA ACATTGCTGA (SEQ IDNO:3), CTCCTGGCAG ACTGGTCAGC (SEQ ID NO:5), TTCCGGAAGC AGGAGAGCTG (SEQID NO:6), and TCCTGGGAAT ACTGGCACGG (SEQ ID NO:7).
 8. The inhibitor ofclaim 7, wherein said antisense oligonucleotide has a nucleotidesequence CTCACAAACA ACATTGCTGA (SEQ ID NO:3).
 9. The inhibitor of claim7, wherein said antisense oligonucleotide has a nucleotide sequenceCTCCTGGCAG ACTGGTCAGC (SEQ ID NO:5).
 10. The inhibitor of claim 7,wherein said antisense oligonucleotide has a nucleotide sequenceTTCCGGAAGC AGGAGAGCTG (SEQ ID NO:6).
 11. The inhibitor of claim 7,wherein said antisense oligonucleotide has a nucleotide sequenceTCCTGGGAAT ACTGGCACGG (SEQ ID NO:7).
 12. An optionally modifiedoligonucleotide for inhibiting expression of human IL-6 receptor,wherein said oligonucleotide is up to 30 nucleotides in length andincludes a nucleotide sequence selected from the group consisting ofCTCACAAACA ACATTGCTGA (SEQ ID NO:3), CTCCTGGCAG ACTGGTCAGC (SEQ IDNO:5), TTCCGGAAGC AGGAGAGCTG (SEQ ID NO:6), and TCCTGGGAAT ACTGGCACGG(SEQ ID NO:7).
 13. The oligonucleotide of claim 12, wherein saidoligonucleotide is modified with a member selected from the groupconsisting of alkylphosphonate, phosphorothioate, phosphorothioate,phosphoroamidate and phosphate triester.
 14. The oligonucleotide ofclaim 13, wherein said oligonucleotide is modified with analkylphosphonate.
 15. The oligonucleotide of claim 13, wherein saidoligonucleotide is modified with a phosphorothioate.
 16. Theoligonucleotide of claim 13, wherein said oligonucleotide is modifiedwith a phosphorodithioate.
 17. The oligonucleotide of claim 13, whereinsaid oligonucleotide is modified with a phosphoroamidate.
 18. Theoligonucleotide of claim 13, wherein said oligonucleotide is modifiedwith a phosphate triester.
 19. The oligonucleotide of claim 14, whereinsaid alkylphosphonate is a methylphosphonate.
 20. The oligonucleotide ofclaim 14, wherein said alkylphosphonate is an ethylphosphonate.
 21. Amethod for inhibiting expression of human IL-6 receptor comprising: a)providing to a human cell the oligonucleotide of claim 12; and b)contacting said cell with said oligonucleotide under conditions suchthat said oligonucleotide is delivered into said cell and hybridizeswith a nucleic acid sequence encoding the IL-6 receptor of said cell, sothat the human IL-6 receptor of said cell is inhibited.